Development of Novel Sanitizers for Fresh Vegetables

S. Ahmed1, M. L. Bari1, M. A. Rahman2, M. A. Goffar2, A.L. Acedo Jr.3, W. Easdown3,

J.A. Hughes4 and J.D.H. Keatinge4
1
Center for Advanced Research in Sciences, University of Dhaka, Bangladesh
2
Horticultural Research Center, BARI, Gazipur, Bangladesh
3
AVRDC – The World Vegetable Center South Asia, Hyderabad, India
4
AVRDC – The World Vegetable Center Headquarters, Shanhua, Tainan, Taiwan

Keywords: Fruits and vegetables, pathogens, scallop powder, EO water and ASC

Abstract
Postharvest sanitization of fruit and vegetables is important in enhancing
quality and food safety. In this study, five non-chlorine sanitizers; electrolyzed water
(EO), electrolyzed alkaline water (EOAl), acidified sodium chlorite (ASC), hydrogen
peroxide (H2O2) and scallop powder solution (SP), were evaluated for their
effectiveness in reducing resident bacteria and inoculated pathogen (Escherichia coli
O157:H7) on tomato and eggplant and compared with 200 ppm chlorine solution
and distilled water (control). Tomatoes had high prevalence of total viable bacteria
(5.5 log CFU/g) and coliform (3.3 log CFU/g), and were detected to contain E. coli
(2.4 log CFU/g), Staphylococcus spp. (3.8 log CFU/g), Listeria spp. (3.6 log CFU/g)
and Yersinia spp. (1.7 log CFU/g). Almost similar microbial profile was obtained on
eggplants. No Salmonella spp. and Vibrio spp. was detected on both vegetables.
Washing tomato and eggplant in distilled water removed some soil and other debris
but did not markedly reduce the bacterial load. The different sanitizers reduced the
total viable bacteria by 2.3-3.5 log CFU/g and coliform bacteria by 1.1-1.9 log CFU/g
on tomatoes. The resident pathogenic bacteria were also reduced to below detection
limit by H2O2, ASC and SP. The different sanitizers had similar effect on total viable
and coliform bacteria load on eggplant. However, only H 2O2 and SP reduced the
pathogenic bacteria to below detection limit. On inoculated produce, E. coli
O157:H7 was higher on tomato (5.4 log CFU/g) than on eggplant (4.7 log CFU/g).
Chorine, EO, H2O2 and SP had more potent effect on tomatoes than the other
sanitizers. On eggplant, the pathogen was undetected in all sanitizing treatments,
except EOAl. The results showed that SP and H 2O2 were more promising
alternatives to chlorine that could be applied in fresh produce industries.

INTRODUCTION
Fresh vegetables and fruit have limited shelf life due to their susceptibility to
microbial decay and are prone to contamination by pathogenic microorganisms that cause
human diseases. Fresh produce decontamination is an important step in reducing
microbial population and enhancing quality and food safety. Aycicek et al (2006) quoted
the Hazard Analysis Critical Control Points-Total Quality Management (HACCP-TQM)
technical guidelines qualifying raw foods with less than 4.0 log CFU/g aerobic plate count
as “good”, 4.0-6.7 as “average”, 6.7-7.7 as “poor”, and more than 7.7 as “spoiled”. In
general, raw foods such as vegetables and fruit are considered safe for human
consumption when aerobic plate count is below 4.0 log CFU/g.
Chlorine is the most widely used in produce decontamination. A serious concern
is that chlorine reacts with the organic matter in the produce to form highly carcinogenic

1
trihalomethanes. Chlorine has also been found to be ineffective in some applications.
Other treatments have been studied to decontaminate fresh produce. Some examples are
sodium hypochlorite (NaOCl), acidified NaOCl, calcium hypochlorite [Ca(OCl) 2],
acidified Ca(OCl)2 (Annous et al., 2001; Mahmuda et al., 2008; Inatsu et al., 2010; Elano
et al., 2011), Na3PO4, Tsunami™, Vortexx™, H2O2 (Bari et al., 2005), calcinated calcium
(Islam et al., 2015), gaseous acetic acid (Bari et al., 2005; Ukuku et al., 2005), organic
acid ozone (Inatsu et al., 2011), ozonated water (Kim et al., 2006), electrolyzed water
(Koseki et al., 2001; Bari et al., 2003; Inatsu et al., 2007), acidified electrolyzed water
(Haq et al., 2005; Laíd et al., 2005; Rahman et al., 2010), peroxyacetic acid (Beuchat,
1998; Beuchat et al., 2004), and combination of EDTA and organic acid (Bari et al.,
2005). However, most of these sanitizers are prepared from the dilution of concentrated
solutions or powders, the handling of which involves some risks. Natural and less
hazardous alternatives are preferable and should be simple and economical to apply
(Sawai et al., 2001; Inatsu et al., 2005. In this study, we evaluated the effectiveness of five
non-chlorine sanitizers for tomato and eggplant decontamination; electrolyzed water
(EO), electrolyzed alkaline water (EOAl), acidified sodium chlorite (ASC), hydrogen
peroxide (H2O2), and scallop powder solution (SP).

MATERIALS AND METHODS

Sample Collection
Tomatoes and eggplants were collected from a commercial farm at Norshindi, 50
km from Dhaka City, and immediately used on the same day of collection. Each tomato
weighed 80 ± 0.5g while eggplant, 76 ± 0.5 g. Fruit with defects were discarded.

Washing Protocol
All chemicals used for the preparation of the different washing solutions were
purchased from Wako Pure Chemical Co. Ltd. (Osaka, Japan). Washing solutions were
prepared immediately prior to application and used within 30 min after preparation. The
chlorine solution was prepared using sodium hypochlorite solution at 200 ppm. ASC was
prepared using 5 mM of citric acid and 0.5 g/L of sodium chlorite. EO and EOAl were
generated using ROX-20TA EO generator and sodium chloride (0.1%). The pH of EO
and EOAl was 2.7± 0.1 and 11.2 ± 0.2, respectively. H2O2 at 1.0% solution was prepared
using the commercial 30% H2O2. SP was prepared as 0.01% solution; pH of the SP
solution was 11.4 ± 0.2. The pH was determined using a pH meter (HORIBA Ltd, Tokyo,
Japan) while free available chlorine, using Chlorine Ultra High Range Meter (HI 95771,
Hanna Instruments, Ann Arbor, MI, USA).
For each experiment, one kg samples of tomato and eggplant was washed
separately with 3.0 L of the washing solution in a sterile glass beaker (8 L). Washing was
carried out for 90 seconds at room temperature with hand rubbing. After washing, the
solution was decanted; a second wash was done with 3.0 L of sterile distilled water to
remove the residual washing solution. The washed samples were placed on a sterile
perforated tray in a laminar flow biosafety cabinet for 4h at 22 ± 20C to facilitate drying.

Inoculation Study
Strains of E. coli O157:H7 (CARS-3 isolated from bovine feces) were used. To
minimize the growth of microorganisms naturally present on vegetable samples, all test
strains were adapted to grow in tryptic soy broth (pH 7.3; Oxoid) supplemented with

2
rifampicin (50 µg/ml). Plating on media containing rifampicin greatly minimized
interference with colony development by naturally occurring microorganisms and
facilitate the detection of test pathogen on recovery media. Introduction of drug resistant
mutations into test strains has been used effectively to detect inoculated bacteria in food
products (Inatsu et al., 2007).
E. coli O157:H7 was cultured at 37°C in 5 ml of tryptic soy broth (TSB; oxoid)
medium supplemented with 50 µg/ml rifampicin (TSB-Rif). Cultures were transferred to
TSB-Rif by loop at three successive 24-h intervals immediately before they were used for
inoculation. Cells were collected by centrifugation (3000 rpm, 10 min, at 22°C) and re-
suspended in 9 mL of sterile 0.85% sodium chloride. The inoculums (9 ml) with an initial
concentration of approximately 107 CFU/ml was maintained at 220C ± 10C and applied on
the vegetable samples within 1 hour of preparation. Dip inoculation method was used.
Five hundred grams of each vegetable were dipped into 2.0 L of E. coli O157:H7
inoculum in a beaker and mixed gently with a sterile glass rod for 5 min. This process was
done inside the laminar flow biosafety cabinet. After the inoculum was decanted, samples
were placed on a sterile perforated tray in a laminar flow biosafety cabinet for 4h at (22 ±
2)0 C to facilitate drying. After drying, the washing protocol described above was done.

Microbiological Analysis
Twenty five grams of each vegetable samples were placed in a stomacher bag with
225 mL of sterile saline water. The mixture was pummeled for 90 s and serial decimal
dilutions were prepared with sterile saline water. The diluted and undiluted samples (0.1
mL) were then surface plated on both selective and nonselective agar media. Tryptic Soy
Agar (TSA; Oxoid) medium was used to determine the total bacterial count. Selective
media were used; Sorbitol MacConkey (SMAC) agar for E. coli, Chromogenic agar for
coliform, Bismuth Sulfite Agar (BSA) for Salmonella spp., Cefexime Tellurite-Sorbitol
MacConkey (CT-SMAC) for E. coli O157:H7, Listeria selective agar for Listeria
monocytogens, and Yersinia selective agar for Yersinia spp. In addition, Tryptic soy agar
(TSA; Nussui Co Ltd, Tokyo, Japan) supplemented with 50 µg/ml rifampicin was used as
a nonselective medium for determination of inoculated E. coli O157:H7 study. Samples
containing E. coli O157:H7 were surface-plated onto Sorbitol MacConkey agar
supplemented with cefixime (0.05 mg/L) (CT-SMAC) and potassium tellurite (2.5 mg/L)
(CT-selective supplement, Oxoid) and 50 µg/ml rifampicin. All ingredients except CT-
selective supplement and rifampicin were combined and sterilized by heating at 121 0C for
15 min. The CT-selective supplement and rifampicin were added to the molten agar
before pouring the medium into petri plates.

Statistical Analysis
All trials were replicated three times. Reported plate count data represented the
mean values obtained from three individual trials, with each of these values being
obtained from duplicate samples. Data were subjected to analysis of variance using the
Microsoft Excel program (Redmond, Washington DC, USA.). Significant treatment
effects were separated by the least-significant difference (LSD) test.

RESULTS AND DISCUSSION

High prevalence of total viable bacteria (5.5 log CFU/g) and coliform (3.3 log
CFU/g), and presence of E. coli (2.4 log CFU/g), Staphylococcus spp. (3.8 log CFU/g),
Listeria spp. (3.6 log CFU/g) and Yersinia spp. (1.7 log CFU/g) were recorded in tomato

3
(Table 1). Eggplant had relatively lower prevalence of total viable bacteria (5.2 log
CFU/g), coliform (3.1 log CFU/g), and E. coli (2.2 log CFU/g), but had similar
population of Staphylococcus spp., Listeria spp. and Yersinia spp. (Table 2). No
Salmonella spp. and Vibrio spp. was detected in both vegetables. Washing tomato and
eggplant in distilled water removed some soil and other debris but did not markedly
reduce the bacterial load. The different sanitizers reduced the total viable bacteria by 2.3-
3.5 log CFU/g and coliform bacteria by 1.1-1.9 log CFU/g on tomatoes (Table 1). The
pathogenic bacteria detected (E. coli, Listeria spp., Staphylococcus spp. and Yersinia
spp.) were also reduced to below detection limit by H2O2, ASC and SP treatments.
Listeria spp. was still detected in chlorine, EO and EOAl treatments while
Staphylococcus spp. was detected in chlorine and EO treatments. On eggplant, the
different sanitizers had similar effect on total viable and coliform bacteria load (Table 2).
However, only H2O2 and SP reduced the pathogenic bacteria to below detection limit.
Listeria spp. and Staphylococcus spp. were detected in chlorine, EO and EOAl treatments
while Staphylococcus spp. in ASC treatment. E. coli and Yersinia spp. were reduced to
below detection limit by all sanitizers.
On inoculated produce, E. coli O157:H7 was higher on tomato (5.4 log CFU/g)
than on eggplant (4.7 log CFU/g) (Tables 1-2). Water wash slightly reduced the pathogen
load while the different sanitizers had remarkable reducing effect. Chorine, EO, H 2O2 and
SP had more potent effect on tomatoes as the pathogen load was reduced to below
detection limit. On eggplant, the pathogen was undetected in all sanitizing treatments,
except EOAl. However, after enrichment study, E. coli O157:H7 was detected in all
sanitizing treatments. This result suggests that the sanitizers were only able to reduce the
pathogen load and did not totally eliminate the pathogen.
These results imply that fresh tomato and eggplant from commercial farms were
heavily contaminated with bacteria, which is a serious concern after harvest and during
consumption. All sanitizers were effective in reducing total bacterial load on tomato and
eggplant to below 4.0 log CFU/g which is considered to be the safe level for human
consumption (Aycicek et al., 2006). Of the different sanitizers, H2O2 and SP appeared to
be the more promising as they consistently reduced the total viable and coliform bacteria
and rendered the pathogenic bacteria undetectable on both tomato and eggplant. Between
these two promising sanitizers, SP would be more advantageous being a natural product
which is safe, biodegradable and environment-friendly and will contribute to management
of food industry waste such as scallop shells. SP is prepared by baking the inner portion
of the scallop (Patinopecten yessoensis) shell at 200˚C followed by exposure to heat
treatment at 1000°C. The resulting powder is then passed through a microsieve to obtain
≤15 µm particle sized powder. The powder has bactericidal action due to calcium oxide
that is converted by heat treatment from calcium carbonate, which is the main component
of the shell powder. SP is also known as calcinated calcium.

ACKNOWLEDGEMENT
This research was made possible through the support provided by the Bureau for
Food Security, U.S. Agency for International Development (USAID), under the terms of
Award No. AID-BFS-IO-12-00004. All opinions expressed in this paper are those of the
authors and do not necessarily reflect the views of the USAID.

Literature cited

4
Annous, B.A., Sapers, G.M. and Mattrazzo, A.M. 2001. Efficacy of washing with a
commercial flat-bed brush washer, using conventional and experimental washing
agents, in reducing populations of Escherichia coli on artificially inoculated apples. J.
Food Prot. 64: 159-163.
Aycicek, H., Oguz, U. and Karci, K. 2006. Determination of total aerobic and indicator
bacteria on some raw eaten vegetables from wholesalers in Ankara, Turkey. Intl. J.
Hyg. Envl. Health 209(2): 197–201.
Bari, M.L., Sabina, Y., Isobe, S., Uemura, T. and Isshiki, K. 2003. Effectiveness of
electrolyzed acidic water in killing E. coli O157:H7, Salmonella and Listeria
monocytogenes on the surface of tomatoes. J. Food Prot. 66 (4): 542-548.
Bari, M.L., Ukuku, D.O., Kawasaki, T., Inatsu, Y., Isshiki, K. and Kawamoto, S. 2005.
Combined efficacy of nisin and pediocin with sodium lactate, citric acid, phytic acid
and potassium sorbate and EDTA in reducing Listeria monocytogenes population of
inoculated fresh-cut produce. J. Food Prot. 68 (7): 1381-1387.
Beuchat, L.R. 1998. Surface decontamination of fruits and vegetables eaten raw: A
review. Food Safety Unit, World Health Organization, WHO/FSF/FOS/98.2.
Beuchat, L.R., Alder, B.B. and Lang, M.M. 2004. Efficacy of chlorine and a peroxyacetic
acid sanitizer in killing Listeria monocytogenes on iceberg and romaine lettuce using
simulated commercial processing conditions. J. Food Prot. 67 (6): 1238-1242.
Elano, R.R., Kitagawa, T., Bari, M.L., Kawasaki, S., Kawamoto, S. and Inatsu, Y. 2011.
Comparison of the effectiveness of acidified sodium chlorite and sodium hypochlorite
in reducing Escherichia coli. Foodborne Pathog. Dis. 7 (12): 1481-1489.
Haq, M.I., Sugiyama, J. and Isobe, S. 2005. Applications of electrolyzed water in
agriculture and food industries. Food Sci. Technol. Res. 11 (2): 135-150.
Inatsu, Y., Bari, M.L., Kawasaki, S., Isshiki, K. and Kawamoto, S. 2005. Efficacy of
acidified sodium chlorite treatments in reducing Escherichia coli O157:H7 on Chinese
cabbage. J. Food Prot. 68: 251-255.
Inatsu, Y., Bari, M.L. and Kawamoto, S. 2007. Application of acidified sodium chlorite
prewashing treatment to improve the food hygiene of lightly fermented vegetables.
Jpn. Agric. Res. Quart. 41: 17-23.
Inatsu, Y., Kitagawa, T., Bari, M.L., Nei, D., Juneja, V.K. and Kawamoto, S. 2010.
Effectiveness of acidified sodium chlorite and other sanitizers to control Escherichia
coli O157:H7 on tomato surfaces. Foodborne Pathog. Dis. 7 (6): 629-635.
Inatsu, Y., Kitagawa, T., Nakamura, N., Kawasaki, S., Nei, D., Bari, M.L. and
Kawamoto, S. 2011. Effectiveness of stable ozone microbubble water on reducing
bacteria on the surface of selected leafy vegetables. Food Sci. Tech. Res. 17 (16): 479-
483.
Islam, Z., Sultana, S., Rahman, M.M., Rahman, S.R. and Bari, M.L. 2015. Effectiveness
of different sanitizers in inactivating E. coli O157:H7 in tomato and cucumber. J. Food
Nutr. Sci. Special Issue: Food Processing and Food Quality. 3 (1-2): 60-64.
Kim, J.G., Yaptenco, K.F. and Lim, C.H. 2006. Effect of sanitizers on microbial growth
and quality of fresh-cut carrot shreds. Hortic. Environ. Biotechnol. 47 (6): 313-318.
Koseki, S., Yoshida, K., Isobe, S. and Itoh, K. 2001. Decontamination of lettuce using
acidic electrolized water. J. Food Prot. 64 (5): 652-658.
Laíd, P.C., Vivian, R.C., Mercado, M., Díaz, M. and Carrascal, A.K. 2005. Effectiveness
of electrolyzed oxidizing water for inactivating Listeria monocytogenes in lettuce.
Univ. Sci. 10 (1), 97-108.

5
Mahmuda, Y., Bari, M.L., Inatsu, Y. and Kawamoto, S. 2008. Effect of transient
temperature shift-up on the growth of L. monocytogenes on cut-cabbage during
storage. J. Food Sci. Technol. Res. 14 (5): 493-498.
Rahman, S.M.E., Ding, T. and Oh, D.-H. 2010. Effectiveness of low concentration
electrolyzed water to inactivate foodborne pathogens under different environmental
conditions. Int. J. Food Microbiol. 139: 147-153.
Sawai, J., Satoh, M., Horikawa, M., Shiga, H. and Kojima, H. 2001. Heated scallop-shell
powder slurry treatment of shredded cabbage. J. Food Prot. 64: 1579-1583.
Ukuku, D.O., Bari, M.L., Kawamoto, S. and Isshiki, K. 2005. Use of hydrogen peroxide
in combination with nisin, sodium lactate and citric acid for reducing transfer of
bacterial pathogens from whole melon surfaces to fresh-cut pieces. Int. J. Food
Microbiol. 104 (2): 225-233.

6
 Tables:

Table 1 Effects of different sanitizers on resident bacteria and inoculated E. coli O157:H7 (log CFU/g) on fresh tomato.
1
≤1.0 = below detection limit
2
N Pathogen Control Distilled 200 ppm 1% H2O2+ D
Pathogen EO+DW EOAl+DW ASC+DW SP+DW
= origin (No trt) water (DW) Chlorine DW
Total viable bacteria 5.5 ± 0.5 4.8 ± 0.3 2.2 ± 0.1 2.3 ± 0.1 2.7 ± 0.2 2.0 ± 0.1 2.4 ± 0.2 3.2 ± 0.3
Coliform 3.3 ± 0.2 3.0 1.7 ± 0.3 1.9 ± 0.1 2.2 ± 0.2 1.4 ± 0.1 1.5 ± 0.1 2.0 ± 0.2
E. coli 2.4 ± 0.3 1.8 ± 0.3 ≤1.01 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Salmonella spp. ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Natural
Vibrio spp. ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Listeria spp. 3.6 ± 0.2 3.3 ± 0.3 1.2 ± 0.1 1.2 ± 0.2 1.4 ± 0.2 ≤1.0 ≤1.0 ≤1.0
Staphylococcus spp. 3.8 ± 0.3 2.6 ± 0.4 1.8 ± 0.3 1.4 ± 0.2 ≤1.0 ≤1.0 ≤1.0 ≤1.0

Yersinia spp. 1.7 ± 0.1 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0

E. coli O157:H7
5.4 ± 0.3 4.2 ± 0.2 ≤1.0 ≤1.0 1.9 ± 0.3 ≤1.0 2.3 ± 0.2 ≤1.0
(without enrichment)
Inoculated
E. coli O157:H7
ND2 ND +3 + ND + ND +
(with enrichment)
not done
3
+ = present

7
 Table 2 Effects of different sanitizers on resident bacteria and inoculated E. coli O157:H7 (log CFU/g) on fresh eggplant.
1
≤1.0 = below detection limit
Pathogen Control Distilled 200 ppm 1% H2O2
Pathogen EO+DW EOAl+DW ASC+DW SP+DW
origin (No trt.) water (DW) Chlorine + DW
Total viable bacteria 5.2 ± 0.6 4.8± 0.4 2.2± 0.5 2.3± 0.3 2.7 ± 0.6 2.0 ± 0.1 2.4 ± 0.2 3.2 ± 0.2
Coliform 3.1 ± 0.4 3.0 ± 0.2 1.7 ± 0.1 1.9 ± 0.1 2.2 ± 0.3 1.4 ± 0.1 1.5 ± 0.2 2.0 ± 0.1
E. coli 2.2 ± 0.3 1.8 ± 0.2 ≤1.01 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Salmonella spp. ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Natural
Vibrio spp. ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0
Listeria spp. 2.0 ± 0.5 3.3 ± 0.3 1.2 ± 0.4 1.2 ± 0.2 1.4 ± 0.4 ≤1.0 ≤1.0 ≤1.0
Staphylococcus spp. 2.8 ± 0.1 2.6 ± 0.4 1.8 ± 0.4 1.4 ± 0.3 1.2 ± 0.1 ≤1.0 1.2 ≤1.0

Yersinia spp. 2.1 ± 0.2 1.4 ± 0.3 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0 ≤1.0

E. coli O157:H7
4.7 ± 0.2 4.0 ± 0.1 ≤1.0 ≤1.0 1.7 ± 0.4 ≤1.0 ≤1.0 ≤1.0
(without enrichment)
Inoculated
E. coli O157:H7
ND ND + + + + + +
(with enrichment)
2
ND = not done
3
+ = present